Phophylactic and therapeutic use of hydroxysteroids

ABSTRACT

3-Hydroxy-7-hydroxy steroids and 3-oxo-7-hydroxy steroids, especially the 7β-isomers thereof, and pharmaceutically acceptable esters thereof are useful for protection against ischaemia-induced damage to peripheral organs, such as the heart or kidneys, as well as treatment of spiral cord injury.

[0001] The present invention relates to certain new prophylactic andtherapeutic uses, of 3-hydroxy-7-hydroxy-steroid compounds and ofcertain ketone derivatives thereof, and specifically to the use of thesecompounds for the prevention or treatment of damage caused by ischaemicstress of peripheral organs, such as the heart or the kidneys, as wellas treatment of spinal cord injury.

[0002] Using a specific model for neuroprotection, we have demonstratedthat compounds of this type have neuroprotective activity. We have nowdiscovered that the same mode of action that results in thisneuroprotective effect also operates in the tissues of peripheralorgans, such as the heart and kidneys, and so the compounds have acardioprotective effect and the ability to protect against ischaemicrenal damage also.

[0003] Thus, the present invention provides the use of a3-hydroxy-7-hydroxy steroid or a 3-oxo-7-hydroxy steroid or apharmaceutically acceptable ester thereof for the manufacture of amedicament for protection against ischaemic damage to tissues ofperipheral organs (i.e. any functional tissue in the body except thebrain and the spinal cord), especially cardiac or renal damage, and forthe treatment of spinal cord injury-induced damage to the spinal cord.

[0004] A particular class of compounds which are preferred for use inthe present invention are the 7β-hydroxy steroids, and, of these, thecompounds which are of especial interest to the present invention arethe 3β, 7β-dihydroxy steroids and pharmaceutically acceptable estersthereof.

[0005] Preferred esters are carboxylic acid and amino acid esters,

[0006] Examples of optionally substituted 3β, 7β-dihydroxy steroids andpharmaceutically acceptable esters and other derivatives thereof whichmay be used in the present invention are those compounds of formula (I):

[0007] wherein

[0008] R¹ and R² are the same as or different from each other and eachrepresents a hydrogen atom, an alkyl group having from 1 to 6 carbonatoms, an alkenyl group having from 2 to 6 carbon atoms, an alkynylgroup having from 2 to 6 carbon atoms, an aryl group having from 6 to 10carbon atoms, a formyl group, an alkylcarbonyl group having from 2 to 7carbon atoms, an alkenylcarbonyl group having from 3 to 7 carbon atoms,an alkynylcarbonyl group having from 3 to 7 carbon atoms, anarylcarbonyl group having from 7 to 11 carbon atoms, an aralkylcarbonylgroup having from 8 to 15 carbon atoms, an aralkenylcarbonyl grouphaving from 9 to 15 carbon atoms, a residue of an amino acid, or aheterocyclic-carbonyl group, as defined below;

[0009] one of R^(a) and R^(b) represents a group of formula —R^(c),preferably in the β configuration, and the other represents a hydrogenatom, or R^(a) and R^(b) together represent an oxo group;

[0010] R^(c) represents an alkanoyl group having from 1 to 6 carbonatoms, an aryl-carbonyl group, in which the aryl part is an aromaticcarbocyclic group having from 6 to 10 ring carbon atoms, aheterocyclic-carbonyl group, as defined below, or a group of formula—OR⁴, where R⁴ represents any one of the groups and atoms defined abovefor R¹ and R²;

[0011] the ring A,

[0012] is a benzene or cyclohexane ring;

[0013] when ring A is a cyclohexane ring, the dotted line in ring Brepresents a single or double carbon-carbon bond, and n is 1; or whenring A is a benzene ring, the dotted line in ring B represents a singlecarbon-carbon bond and n is 0;

[0014] said heterocyclic-carbonyl group is a group of formula R³—CO,where R³ represents a heterocyclic group having from 3 to 7 ring atoms,of which from 1 to 3 are hetero-atoms selected from nitrogen atoms,oxygen atoms and sulphur atoms, and the remaining atom or atoms of whichthere is at least one is or are carbon atoms;

[0015] said alkyl, alkenyl and alkynyl groups and the alkyl alkenyl andalkynyl parts of said alkylcarbonyl, alkenylcarbonyl and alkynylcarbonylgroups being unsubstituted or having at least one of the followingsubstituents Ψ:

[0016] substituents Ψ: hydroxy groups, mercapto groups, halogen atoms,amino groups, alkylamino groups having from 1 to 6 carbon atoms,dialkylamino groups in which each alkyl group has from 1 to 6 carbonatoms, carbamoyl groups, nitro groups, alkoxy groups having from 1 to 6carbon atoms, alkylthio groups having from 1 to 6 carbon atoms, carboxygroups, alkoxycarbonyl groups and unsubstituted aryl groups having from6 to 10 carbon atoms;

[0017] said aryl groups, said heterocyclic groups, and the aryl parts ofsaid arylcarbonyl groups and said aralkylcarbonyl groups beingunsubstituted or having at least one of the following substituents ξ:

[0018] substituents ξ: any of substituents Ψ, and alkyl groups havingfrom 1 to 6 carbon atoms, hydroxyalkyl groups having from 1 to 6 carbonatoms, and haloalkyl groups having from 1 to 6 carbon atoms;

[0019] and pharmaceutically acceptable salts and esters thereof.

[0020] The activity of the compounds of the present invention isillustrated by the accompanying drawings, in which:

[0021]FIG. 1A shows data relating to Example 22 presented as mean number±SEM of intact neurons per 400 μm length of CA1 region;

[0022]FIG. 1B shows data of Example 22 expressed as percentage of intactneurons per 400 μm length of CA1 region compared to sham operatedanimals set as 100%;

[0023]FIG. 1C shows data of Example 22 presented as absolute percentageof neuroprotection when the number of surviving neurons in the ischaemiagroup was set to zero and those of the sham operated group was set to100%;

[0024]FIG. 2 shows infarct size in Example 23 in control vehicle treatedhearts and 7β-OH EPIA treated hearts before, during and after regionalischaemia; 7β-OH EPIA was added to the perfusate at 25 minutes, regionalischaemia was introduced at 55 minutes, the ischaemic area wasreperfused at 85 minutes;

[0025]FIG. 3 shows coronary flow in Example 23 in control vehicletreated hearts and 7β-OH EPIA treated hearts before, during and afterregional ischaemia; 7β-OH EPIA was added to the perfusate at 25 minutes,regional ischaemia was introduced at 55 minutes, the ischaemic area wasreperfused at 85 minutes;

[0026]FIG. 4 shows heart rate in Example 23 in control vehicle treatedhearts and 7β-OH EPIA treated hearts before, during and after regionalischaemia; 7β-OH EPIA was added to the perfusate at 25 minutes, regionalischaemia was introduced at 55 minutes, the ischaemic area wasreperfused at 85 minutes;

[0027]FIG. 5 shows the left ventricular developed pressure in Example 23in control vehicle treated hearts and 7β-OH EPIA treated hearts before,during and after regional ischaemia; 7β-OH EPIA was added to theperfusate at 25 minutes, regional ischaemia was introduced at 55minutes, the ischaemic area was reperfused at 85 minutes; and

[0028]FIG. 6 shows the end diastolic pressure in Example 23 in controlvehicle treated hearts and 7β-OH EPIA treated hearts before, during andafter regional ischaemia; 7β-OH EPIA was added to the perfusate at 25minutes, regional ischaemia was introduced at 55 minutes, the ischaemicarea was reperfused at 85 minutes.

[0029] In the above compounds of formula (1), the group —OR² at the7-position may be in the α or β configuration, but is preferably in theβ configuration.

[0030] More preferably, in the compounds of formula (1):

[0031] R¹ and R² are the same as or different from each other and eachrepresents a hydrogen atom, an alkyl group having from 1 to 6 carbonatoms, an optionally substituted phenyl group, a formyl group, analkylcarbonyl group having from 2 to 5 carbon atoms, an arylcarbonylgroup having from 7 to 11 carbon atoms, an aralkylcarbonyl group havingfrom 8 to 15 carbon atoms, a residue of an amino acid, or aheterocyclic-carbonyl group, as defined below; we particularly preferthat R¹ and R² should both represent hydrogen atoms;

[0032] one of R^(a) and R^(b) represents an alkanoyl group having from 1to 6 carbon atoms or a group of formula —OR⁴, where R⁴ represents anyone of the groups and atoms defined above for R¹ and R², preferably inthe β configuration, and the other represents a hydrogen atom, or R^(a)and R^(b) together represent an oxo group; we particularly prefer thatR^(a) and R^(b) should together represent an oxo group, or that one ofR^(a) and R^(b) should represent a hydrogen atom and the other shouldrepresent a hydroxy group or an alkanoyl group having from 1 to 4 carbonatoms, especially a hydroxy group or an acetyl group;

[0033] said heterocyclic-carbonyl group is a group of formula R³—CO,where R³ represents a heterocyclic group-having from 3 to 7 ring atoms,of which from 1 to 3 are hetero-atoms selected from nitrogen atoms,oxygen atoms and sulphur atoms, and the remaining atom or atoms of whichthere is at least one is or are carbon atoms.

[0034] Most preferred compounds of formula (I) are those compounds inwhich:

[0035] R¹ and R² both represent hydrogen atoms; and

[0036] R^(a) and R^(b) together represent an oxo group, or one of R^(a)and R^(b) represents a hydrogen atom and the other represents a hydroxygroup or an alkanoyl group having from 1 to 4 carbon atoms, especially ahydroxy group or an acetyl group;

[0037] and pharmaceutically acceptable esters thereof.

[0038] Examples of 3-oxo-7β-hydroxy steroids which may be used in thepresent invention are those compounds of formula (II):

[0039] in which R^(a), R^(b) and R² are as defined above, and preferablyR^(a) and R^(b) together represent an oxo group, or one of R^(a) andR^(b) represents a hydrogen atom and the other represents a hydroxygroup, preferably in the β-configuration, or an acetyl group;

[0040] and pharmaceutically acceptable esters thereof.

[0041] In the above compounds of formula (II), the group —OR² at the7-position may be in the α or β configuration, but is preferably in theβ configuration.

[0042] More preferably, in the compounds of formula (II):

[0043] R² represents a hydrogen atom, an alkyl group having from 1 to 6carbon atoms, an optionally substituted phenyl group, a formyl group, analkylcarbonyl group having from 2 to 5 carbon atoms, an arylcarbonylgroup having from 7 to 11 carbon atoms, an aralkylcarbonyl group havingfrom 8 to 15 carbon atoms, or a heterocyclic-carbonyl group, as definedbelow; we particularly prefer that R² should represent a hydrogen atom;

[0044] one of R^(a) and R^(b) represents an alkanoyl group having from 1to 6 carbon atoms or a group of formula —OR⁴, where R⁴ represents anyone of the groups and atoms defined above for R², preferably in the βconfiguration, and the other represents a hydrogen atom, or R^(a) andR^(b) together represent an oxo group; we particularly prefer that R^(a)and R^(b) should together represent an oxo group, or that one of R^(a)and R^(b) should represent a hydrogen atom and the other shouldrepresent a hydroxy group or an alkanoyl group having from 1 to 4 carbonatoms, especially a hydroxy group or an acetyl group;

[0045] said heterocyclic-carbonyl group is a group of formula R³—CO,where R³ represents a heterocyclic group having from 3 to 7 ring atoms,of which from 1 to 3 are hetero-atoms selected from nitrogen atoms,oxygen atoms and sulphur atoms, and the remaining atom or atoms of whichthere is at least one is or are carbon atoms;

[0046] and pharmaceutically acceptable esters thereof.

[0047] Most preferred compounds of formula (II) are those compounds inwhich:

[0048] R² represents a hydrogen atom; and

[0049] R^(a) and R^(b) together represent an oxo group, or one of R^(a)and R^(b) represents a hydrogen atom and the other represents a hydroxygroup or an alkanoyl group having from 1 to 4 carbon atoms, especially ahydroxy group or an acetyl group;

[0050] and pharmaceutically acceptable esters thereof.

[0051] In the compounds of the present invention, where R¹, R², R⁴ orsubstituent ξis an alkyl group, this may be a straight or branched chainalkyl group having from 1 to 6 carbon atoms, and examples include themethyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl,pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1-ethylpropyl,2-ethylpropyl, 1,1-dimethylpropyl, hexyl, 1-methylpentyl,2-methyl-pentyl, 3-methylpentyl, 4-methylpentyl, 1-ethylbutyl,2-ethylbutyl, 3-ethylbutyl, t-hexyl, and 1,1-dimethylpentyl groups, ofwhich those groups having from 1 to 4 carbon atoms are preferred, themethyl and ethyl groups being most preferred.

[0052] Where R¹, R² or R⁴ represents an alkenyl group, this may be astraight or branched chain alkenyl group having from 2 to 6 carbonatoms, and examples include the vinyl, 1-propenyl, allyl, isopropenyl,methallyl, 1-, 2-, 3-butenyl, isobutenyl, 1-, 2-, 3-, 4-pentenyl and 1-,2-, 3-, 4-, 5-hexenyl groups, of which those alkenyl groups having from2 to 4 carbon atoms-are preferred, the vinyl and allyl groups being mostpreferred.

[0053] Where R¹, R² or R⁴ represents an alkynyl group, this may be astraight or branched chain alkynyl group having from 2 to 6 carbonatoms, and examples include the ethynyl, 1-, 2-propynyl, 1-, 2-,3-butynyl, isobutynyl, 1-, 2-, 3-, 4-pentynyl and 1-, 2-, 3-, 4-,5-hexynyl groups, of which those alkynyl groups having from 2 to 4carbon atoms are preferred.

[0054] Where R¹, R², R⁴ or substituent Ψ represents an aryl group, thisis an aromatic carbocyclic group having from 6 to 10 carbon atoms.Examples of such groups include the phenyl, 1-naphthyl, 2-naphthyl andindenyl groups, of which the phenyl group is preferred. Except in thecase of substituent Ψ, these groups may be substituted or unsubstituted.Where the group is substituted, the number of substituents is limitedonly by the number of substitutable positions, and possibly, in someinstances, by steric constraints. Thus, in the case of the phenylgroups, the maximum number of substituents is 5, in the case of thenaphthyl groups, the maximum number of substituents is 7 and so on.However, a preferred number of substituents is from 1 to 3, and thesubstituents are as hereafter described.

[0055] Where R¹, R² or R⁴ represents an alkylcarbonyl group, this is analkanoyl group, which may be a straight or branched chain group havingfrom 2 to 7 carbon atoms (i.e. from 1 to 6 carbon atoms in the alkylpart), and examples include the acetyl, propionyl, butyryl, isobutyryl,valeryl, isovaleryl, pivaloyl, hexanoyl, and heptanoyl groups, of whichthose groups having from 2 to 5 carbon atoms are preferred, the acetyland propionyl groups being most preferred. The alkyl portion of thisgroup may be substituted or unsubstituted, and, if substituted, thesubstituents are selected from substituents Ψ. Examples of suchsubstituted groups include the alanyl, β-alanyl phenylalanyl,asparaginyl cysteinyl, glycoloyl, glycyl, methionyl, ornithyl,glyceroyl, tropoyl, glutaminyl, glutamyl, homocysteinyl, seryl,homoseryl, threonyl, lactoyl, leucyl, isoleucyl, norleucyl, lysyl,valyl, norvalyl and sarcosyl groups.

[0056] Where R¹, R² or R⁴ represents an alkenylcarbonyl group, this maybe a straight or branched chain alkenylcarbonyl group having from 3 to 7carbon atoms, and examples include the acryloyl, methacryloyl,crotonoyl, isocrotonoyl, 3-butenoyl, pentenoyl and hexenoyl groups, ofwhich those alkenylcarbonyl groups having from 3 to 5 carbon atoms arepreferred, the acryloyl and methacryloyl groups being most preferred.

[0057] Where R¹, R² or R⁴ represents an alkynylcarbonyl group, this maybe a straight or branched chain alkynylcarbonyl group having from 3 to 7carbon atoms, and examples include the propioloyl, 3-butynylcarbonyl,pentynylcarbonyl and hexynylcarbonyl groups, of which thosealkynylcarbonyl groups having from 3 to 5 carbon atoms are preferred.

[0058] Where R^(c), R¹, R² or R⁴ represents an arylcarbonyl group, thearyl part of this may be any of the aryl groups defined and exemplifiedabove. Preferred arylcarbonyl groups include the benzoyl, o-, m- orp-toluoyl, o-, m- or p-anisoyl, o-, m- or p-hydroxybenzoyl, picryl,galloyl, protocatechuoyl, vanilloyl, veratroyl, anthraniloyl,1-naphthoyl and 2-naphthoyl groups.

[0059] Where R¹, R² or R⁴ represents an aralkylcarbonyl oraralkenylcarbonyl group, the aryl and, as the case may be, alkyl oralkenyl group may be any of those groups defined and exemplified above.Specific examples of such groups include the phenylacetyl,3-phenylpropionyl, benziloyl, tyrosyl, atropoyl, hydratropoyl andcinnamoyl groups.

[0060] Where R^(c), R¹, R² or R⁴ represents a heterocyclic-carbonylgroup, this is a group of formula R³—CO—, where R³ represents aheterocyclic group having from 3 to 7 ring atoms, of which from 1 to 3are nitrogen, oxygen or sulphur atoms, the remainder being carbon atoms.At least one of the ring atoms should be a carbon atom. Where there are3 hetero-atoms, it is preferred that at least one is a nitrogen atom.Examples of such groups include the 2- and 3-furoyl, 2- and 3-thenoyl,2-pyridinecarbonyl, nicotinoyl, isonicotinoyl, prolyl,piperidinecarbonyl, piperazinecarbonyl and morpholinocarbonyl groups.

[0061] Where R¹ and/or R² represents a residue of an amino acid, thismay be any amino acid in which a hydroxy group has been removed from theor a carboxy (—COOH) group. Examples of such amino acid residues includethe alanyl, β-alanyl, cystathionyl, cystyl, glycyl, histidyl, homoseryl,isoleucyl, lanthionyl, leucyl, lysyl, methionyl, norleucyl, norvalyl,omithyl, prolyl, sarcosyl, seryl, threonyl, thyronyl, tyrosyl, valylcysteinyl, homocysteinyl, tryptophyl, α-aspartyl, β-aspartyl, aspartoyl,asparaginyl, α-glutamyl, γ-glutamyl, and glutaminyl groups.

[0062] Where R^(c) represents an alkanoyl group, this may be a straightor branched chain group having from 1 to 6 carbon atoms, and examplesinclude the formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl,isovaleryl, pivaloyl, hexanoyl, and heptanoyl groups, of which thosegroups having from 2 to 5 carbon atoms are preferred, the acetyl andpropionyl groups being more preferred, and the acetyl group being mostpreferred.

[0063] Where substituent Ψ or substituent ξ is an alkylamino grouphaving from 1 to 6 carbon atoms, the alkyl part may be any of the alkylgroups defined and exemplified above. Preferred examples of suchalkylamino groups include the methylamino, ethylamino, propylamino,isopropylamino, butylamino, isobutylamino, sec-butylamino, t-butylamino,pentylamino, isopentylamino, neopentylamino, t-pentylamino, hexylamino,and isohexylamino groups, of which those groups having from 1 to 4carbon atoms are preferred, the methylamino and ethylamino groups beingmost preferred.

[0064] Where substituent Ψ or substituent ξ is a dialkylamino group,each alkyl part has from 1 to 6 carbon atoms, and the two alkyl groupsmay be the same as or different from each other. The alkyl groups may beany of the alkyl groups defined and exemplified above. Preferredexamples of such dialkylamino groups include the dimethylamino,methylethylamino, diethylamino, methylpropylamino, dipropylamino,diisopropylamino, ethylbutylamino, dibutylamino, di-t-butylamino,methylpentylamino, dipentylamino, diisopentylamino, and dihexylaminogroups, of which those groups having from 1 to 4 carbon atoms in eachalkyl group are preferred, the dimethylamino and diethylamino groupsbeing most preferred.

[0065] Where substituent Ψ or substituent ξ is an alkoxy group, this maybe a straight or branched chain alkoxy group having from 1 to 6 carbonatoms, and examples include the methoxy, ethoxy, propoxy, isopropoxy,butoxy, isobutoxy, sec-butoxy, t-butoxy, pentyloxy, isopentyloxy,neopentyloxy, t-pentyloxy, hexyloxy, and isohexyloxy groups, of whichthose groups having from 1 to 4 carbon atoms are preferred, the methoxyand ethoxy groups being most preferred.

[0066] Where substituent Ψ or substituent ξ is an alkylthio group havingfrom 1 to 6 carbon atoms, the alkyl part may be any of the alkyl groupsdefined and exemplified above. Preferred examples of such alkylthiogroups include the methylthio, ethylthio, propylthio, isopropylthio,butylthio, isobutylthio, sec-butylthio, t-butylthio, pentylthio,isopentylthio, neopentylthio, t-pentylthio, hexylthio, and isohexylthiogroups, of which those groups having from 1 to 4 carbon atoms arepreferred, the methylthio and ethylthio groups being most preferred.

[0067] Where substituent Ψ or substituent ξ is an alkoxycarbonyl group,this may be a straight or branched chain alkoxycarbonyl group havingfrom 2 to 7 carbon atoms, and examples include the methoxycarbonyl,ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl,isobutoxycarbonyl, sec-butoxycarbonyl, t-butoxycarbonyl,pentyloxycarbonyl, isopentyloxycarbonyl, neopentyloxycarbonyl,t-pentyloxycarbonyl, hexyloxycarbonyl, and isohexyloxycarbonyl groups,of which those groups having from 1 to 4 carbon atoms are preferred, themethoxycarbonyl and ethoxycarbonyl groups being most preferred.

[0068] Where substituent ξ is a hydroxyalkyl group having from 1 to 6carbon atoms, the alkyl part may be any of the alkyl groups defined andexemplified above. Preferred examples of such hydroxyalkyl groupsinclude the hydroxymethyl, 1- and 2-hydroxyethyl, 1-, 2- and3-hydroxypropyl, 1,2-dihydroxyethyl, 1,2,3-trihydroxy-propyl,4-hydroxybutyl, 5-hydroxypentyl and 6-hydroxyhexyl groups.

[0069] Where substituent ξ is a haloalkyl group having from 1 to 6,preferably from 1 to 4, carbon atoms, the alkyl part may be as definedand exemplified above, and the halogen atom is preferably chlorine,fluorine, bromine or iodine. Examples of such groups include thefluoromethyl, chloromethyl, bromomethyl, iodomethyl, dichloromethyl,difluoromethyl, trichloromethyl, triruoromethyl, 2,2,2-trichloroethyl,2-chloroethyl, 2-fluoroethyl, 2-bromoethyl, 2-iodoethyl,2,2-dibromoethyl, 2,2,2-tribromoethyl, 3-fluoropropyl, 3-chloropropyl,4-bromobutyl, 4-fluorobutyl, 5 -fluoropentyl and 6-fluorohexyl groups.

[0070] It will be appreciated that, where the compound contains a groupof formula —OR, where R is any of the groups and atoms defined above inrelation to R¹ etc., the active species is likely to be the compoundcontaining the free hydroxy group. Accordingly, any group that can beconverted in vivo to a hydroxy group may be used in place of the hydroxygroup.

[0071] Specific examples of compounds which may be used in the presentinvention include:

[0072] Of the compounds listed above, the 7β-isomers are preferred.

[0073] Where the compounds of the present invention contain a hydroxygroup, they may be converted to corresponding salts or esters, as iswell known in the art, and there is no particular limitation upon thenature of the salt or ester produced. Where these salts or esters are tobe administered to a patient, then they should be pharmaceuticallyacceptable. However, if the compound is intended for some other purpose,e.g. as an intermediate in another synthesis, then even this restrictionis not necessary. Salts and esters may be chosen from those well knownin the art for this type of compound. Preferred esters are thecarboxylic esters and the amino acid esters, such as, for example, thealanine, β-alanine, cystathionine, cystine, glycine, histidine,homoserine, isoleucine, lanthionine, leucine, lysine, methionine,norleucine, norvaline, ornithine, proline, sarcosine, serine, threonine,thyronine, tyrosine, valine, cysteine, homocysteine, tryptophan,aspartic acid, asparagine, glutamic acid, and glutamine.

[0074] The compounds of the present invention may be prepared by avariety of processes, well known in themselves, starting from the parentsteroids. For example, they may be prepared by the methods described inthe literature referred to above, which would give a mixture of the 7βand corresponding 7α compounds, which may then, if desired, be separatedby well known techniques. However, in some circumstances it may bedesired or convenient to use the mixture of 7α and 7β isomers withoutseparating them.

[0075] As an example, 7α-hydroxy EPIA and 7β-hydroxy EPIA may beobtained from DHEA by allylic oxidation after protection of the3β-hydroxy group and the 17-ketone group using conventional methods. Theproduct is then reduced with a soluble metal compound catalyst (such assodium hydride) and the 3β-hydroxy and 17-ketone groups are deprotected.The 7α-hydroxy and 7β-hydroxy epimers may then be separated byconventional means, for example column chromatography, and the7α-hydroxy EPIA and 7β-hydroxy EPIA may be crystallised to purity.

[0076] An alternative synthetic method is shown in the followingreaction scheme:

[0077] In the above formulae, TBDMSO represents t-butyldimethylsilyloxyand Ac represents acetyl. These abbreviations have the same meaningswhen used hereafter.

[0078] In the first step of the above reaction scheme, the compound offormula (III), oestrone, is protected by a t-butyldimethylsilyloxy groupin a conventional manner to give the protected compound of formula (IV).This is then reacted with ethylene glycol in the presence of an acidcatalyst (such as p-toluenesulphonic acid) to protect the keto group atthe 17 position and give the compound of formula (V). A hydroxy groupmay then be introduced at the 6-position as illustrated hereafter inExample 3, to give the compound of formula (VI), which is thendehydrated to give the compound of formula (VII). This is epoxidised togive the compound of formula (VIII), which is then reduced to thecompound of formula (IX), with a 7α-hydroxy group. Thet-butyldimethylsilyl protecting group is removed, giving the compound offormula (X), and this is heated with a catalytic amount of an acid, togive 7α-hydroxy-oestrone (XI), which may be used in the presentinvention. This may then be oxidised, e.g. using chromic acid/sulphuricacid, to give the 7-keto-oestrone (XI), which is then reacted withacetic anhydride, to give the compound of formula (XIII). This compoundis hydrogenated, e.g. using hydrogen in the presence of a palladiumcatalyst, to give the compound of formula (XIV), and finally the acetylgroups are removed, to give 7β-hydroxy-oestrone (XV), a compound of thepresent invention If desired, this may be reduced, to give7β-hydroxy-oestradiol (XVI), also a compound of the present invention.The corresponding 7α compound may be prepared in an analogous mannerfrom 7α-hydroxy-oestrone (XI).

[0079] Other 7α- and 7β-hydroxy- compounds of the present inventionmaybe prepared in a similar manner, for example, 7β-hydroxy-DHEA may beprepared as illustrated by the following reaction scheme:

[0080] In this reaction scheme, DBEA (XVII) is acetylated to give thecorresponding acetate of formula (XVIII), which is then reacted withethylene glycol, to give the ketal of formula (XIX). The ketal (XIX) isthen oxidised as described in Example 16, to give the corresponding7-keto compound (XX), which is then deacetylated, to give the compoundof formula (XXI). This is reduced, to give 7-hydroxy-17-ketal-EPIA offormula (XXII), which is then treated with an acid to remove the ketalgroup and give 7-hydroxy-EPIA, which is finally separated into the 7β-and 7α- isomers by chromatography, to give 7α-hydroxy-EPIA (XXIV) and7β-hydroxy-EPIA (XXV).

[0081] Each of the steps of the above reaction schemes is individuallywell known and may be carried out using known solvents and catalysts (ifappropriate) and under known reaction conditions, for example conditionsof time and temperature.

[0082] The compounds defined above have a neuroprotective effect. Inaccordance with the present invention, we have found that they also havea cardioprotective effect, and so can be used for the prevention ortreatment of cardiac diseases arising from ischaemic damage, for examplemyocardial infarction. They also have the ability to protect againstischaemic renal damage, for example glomerulonephritis or acute renaldamage. In general, based on the activity demonstrated, it ispredictable that they will have a similar protective effect on tissuesof other peripheral organs.

[0083] Indeed, the compounds of the present invention can also be usedto treat spinal cord injury.

[0084] The compounds of the present invention may be applied to thepatient if it is suspected that they are in danger of an ischaemicevent, especially a myocardial infarction or ischaemic renal damage.Such prophylactic application may be exceedingly useful. However, it hasalso been demonstrated that the compounds of the present invention haveuseful activity, even if applied after an ischaemic event, but it willbe appreciated that it is preferred to administer the compounds as soonas possible, in order to avoid as much myocardial or renal tissue damageas possible. In some circumstances it may be desirable to administerrepeated doses, especially where the patient remains in danger of anischaemic event.

[0085] The compounds may also be administered prophylactically inanticipation of a spinal cord injury or may be used to treat such aninjury after it has occurred.

[0086] Suitable methods of administration are generally by injection, inorder to achieve the desired result as soon as possible. Thus,intravenous injection is particularly preferred.

[0087] The dose of the compound of the present invention will varydepending upon many factors, including the age, body weight and generalcondition of the patient, as well as the mode, frequency and route ofadministration. However, a dose of from 0.01 to 50 mg/kg body weight isgenerally recommended, a dose of from 0.05 to 20 mg/kg body weight beingmore preferred. This may be administered in a single dose or in divideddoses.

[0088] The invention is further illustrated by the followingnon-limiting Examples, of which Examples 1 to 20 illustrate thepreparation of compounds of the present invention and Examples 21 to 23illustrate their activity. In Examples 1 to 20, the Roman numerals referto the formulae in the reaction schemes shown above.

EXAMPLE 1

[0089] 3-t-Butyldimethylsilyl-oestrone (IV)

[0090] 4.25 g of t-butyldimethylsilyl chloride (28.2 mmol, 3 eq.) wereadded to a solution of 50 ml of dimethylformamide (DMF) containing 2.54g of oestrone (III) (9.41 mmol, 1 eq.) and 3.84 g of imidazole (56.5mmol, 6 eq.) in a 100 ml three-necked flask. The mixture was then leftovernight at room temperature under a nitrogen atmosphere. A 10% w/vaqueous potassium carbonate solution was added to the reaction medium,which was then extracted with ethyl acetate. The organic phase waswashed with water and then dried over anhydrous sodium sulphate andevaporated to dryness. 3.76 g of 3-t-butyldimethylsilyl-oestrone(IV)(9.41 mmol, 100%) were obtained.

EXAMPLE 2

[0091] 17-Ketal-3-t-butyldimethylsilyl-oestrone (V)

[0092] A solution of 60 ml of toluene containing 3 g of3-t-butyldimethylsilyl-oestrone (IV)(7.50 mmol), 3 ml of ethylene glycoland a catalytic amount of p-toluenesulphonic acid was heated to refluxwith steam distillation using a Dean-Stark apparatus for 24 hours. Thereaction medium was then poured into 50 ml of a 10% w/v aqueouspotassium carbonate solution. The organic phase was decanted. Theaqueous phase was extracted with ethyl acetate. The organic phases werecombined and evaporated to dryness. 3.16 g of17-ketal-3-t-butyldimethylsilyl-oestrone (V)(7.12 mmol, 95%) wereobtained.

EXAMPLE 3

[0093] 6α-Hydroxy-17-ketal-3-t-butyldimethylsilyl-oestrone (VI)

[0094] In a 1 litre three-necked flask, a solution of 100 ml ofanhydrous tetrahydrofuran (THF) was degassed by nitrogen flushing andcooled to −80° C. Diisopropylamine (20 ml, 143.30 mmol) was added to thereaction medium. A 15% w/v butyllithium solution in cyclohexane (89.9ml, 143.30 mmol) was added dropwise to the reaction medium. After 10minutes, a solution of 100 ml of anhydrous THF, previously degassed,containing 17.5 g of potassium t-butyrate was added dropwise to thereaction medium. After a further 15 minutes, a solution of 50 ml ofanhydrous THF, previously degassed, containing 12.27 g of17-ketal-3-t-butyldimethylsilyl-oestrone (V)(27.63 mmol) was addeddropwise to the reaction medium. The reaction mixture was left for 2hours at −80° C. At the end of this time, 48 ml of trimethyl borate(429.90 mmol) were added dropwise at −80° C. to the reaction medium,which was left at 0° C. for 1 hour. 100 ml of 30% v/v aqueous hydrogenperoxide solution were then added. The reaction mixture was left for 1hour at room temperature and then 500 ml of water were added. Thereaction medium was extracted with ethyl acetate. The organic phase waswashed with a 10% w/v aqueous sodium thiosulphate solution, washed withwater, dried over anhydrous sodium sulphate and evaporated to dryness.The residue was purified by flash chromatography (SiO2/ethyl acetate:cyclohexane {fraction (1/9)} then {fraction (2/8)}). 6.35 g of6α-hydroxy-17-ketal-3-t-butyldimethylsilyl-oestrone (VI)(13.81 mmol,50%) were obtained.

EXAMPLE 4

[0095] 17-Ketal-3-t-butyldimethylsilyl-6-dehydroestrone (VII)

[0096] A solution of 40 ml of toluene containing 1.54 g of6α-hydroxy-17-ketal-3-t-butyldimethylsilyl-oestrone (VI)(3.35 mmol), 4ml of ethylene glycol and a catalytic amount of p-toluenesulphonic acidwas heated to reflux with steam distillation using a Dean-Starkapparatus for 24 hours. The reaction medium was then poured into 50 mlof a 10% w/v aqueous potassium carbonate solution The organic phase wasdecanted. The aqueous phase was then extracted with ethyl acetate. Theorganic phases were combined and evaporated to dryness. 1.48 g of17-ketal-3-t-butyldimethylsilyl-6-dehydroestrone (VII)(3.35 mmol, 100%)were obtained.

EXAMPLE 5

[0097] 17-Ketal-3-t-butyldimethylsilyl-6α, 7α-epoxyoestrone (VIII)

[0098] A solution of 20 ml of dichloromethane containing 1.16 g ofm-chlorobenzoic acid (55%, 3.69 mmol, 1.1 eq.) was added dropwise, at 0°C., to a solution of 20 ml of dichloromethane containing 1.85 g of17-ketal-3-t-butyldimethylsilyl-6-dehydroestrone (VII) (3.36 mmol, 1eq.). The reaction medium was poured, after 2 hours, into a 10% w/vaqueous sodium hydrogen carbonate solution and then extracted with ethylacetate. The organic phase was dried over anhydrous sodium sulphate andthen evaporated to dryness. The residue was purified by flashchromatography (SiO2/ethyl acetate: cyclohexane {fraction (1/9)}). 769mg of 17-ketal-3-t-butyldimethylsilyl-6αc, 7α-epoxyoestrone (VIII)(1.68mmol, 50%) were obtained.

EXAMPLE 6

[0099] 7α-Hydroxy-17-ketal-3-t-butyldimethylsilyl-oestrone (IX)

[0100] 200 mg of lithium aluminium hydride (5.40 mmol, 2 eq.) were addedto a solution of 50 ml of anhydrous THF containing 1.13 g of17-ketal-3-t-butyldimethylsilyl-6α, 7α-epoxyoestrone (VIII)(2.60 mmol, 1eq.). The reaction medium was heated to reflux for 2 hours and thencooled, poured into ice, filtered through a Celite (trade mark) filteraid and extracted with ethyl acetate. The organic phase was dried overanhydrous sodium sulphate and then evaporated to dryness. The residuewas purified by flash chromatography (SiO2/ethyl acetate: cyclohexane{fraction (1/9)}). 837 mg of7α-hydroxy-17-ketal-3-t-butyldiunethylsilyl-oestrone (IX)(1.82 mmol,70%) were obtained.

EXAMPLE 7

[0101] 7α-Hydroxy-17-ketal-oestrone (X)

[0102] A solution of 20 ml of THF containing 1.5 g of tetrabutylammoniumchloride (4.78 mmol, 1.10 eq.) was added, at room temperature, to asolution of 50 ml of THF containing 2 g of7α-hydroxy-17-ketal-3-t-butyldiinethylsilyl-oestrone (IX)(4.35 mmol, 1eq.). The reaction medium was poured into 70 ml of a 10% w/v aqueoussodium carbonate solution. The reaction medium was extracted with ethylacetate. The organic phase was dried over anhydrous sodium sulphate andthen evaporated to dryness. 1.39 g of 7α-hydroxy-17-ketal-oestrone(X)(4.22 mmol, 97%) were obtained.

EXAMPLE 8

[0103] 7α-Hydroxy-oestrone (XI)

[0104] A solution of 50 ml of acetone containing 1 ml of water, 1.0 g of7α-hydroxy-17-ketal-oestrone (X)(3.03 mmol) and a catalytic amount ofp-toluenesulphonic acid was heated to reflux for 2 hours. The reactionmedium was then poured into 70 ml of a 10% w/v aqueous sodium carbonatesolution. The reaction medium was extracted with ethyl acetate. Theorganic phase was dried over anhydrous sodium sulphate and thenevaporated to dryness. 814 mg of 7α-hydroxy-oestrone (XI)(2.85 mmol,94%), which was recrystallised from ethyl acetate, were obtained.

EXAMPLE 9

[0105] 7-Ketoestrone (XII)

[0106] An 8 N solution of chromic acid in sulphuric acid was addeddropwise, until the yellow colour persisted, to a solution cooled to 0°C. of 40 ml of acetone containing 300 mg of 7α-hydroxy-oestrone(XI)(1.05 mmol). The reaction medium was poured into 50 ml of water andthen extracted with ethyl acetate. The organic phase was washed with anaqueous sodium carbonate solution and then dried over anhydrous sodiumsulphate and evaporated to dryness. The residue was purified by flashchromatography (SiO2/ethyl acetate: cyclohexane {fraction (3/7)}). 200mg of 7-keto-estrone (XII)(0.70 mmol, 67%) were obtained.

EXAMPLE 10

[0107] 7-Hydroxy-6-dehydroestrone 3,7-diacetate (XIII)

[0108] A solution of 10 ml of acetic anhydride containing 5 g ofanhydrous sodium acetate and 1 g of 7-keto-oestrone (XII)(3.52 mmol) washeated to reflux for 1 hour. The reaction medium was then cooled andthen poured into water and extracted with diethyl ether. The organicphase was washed with an aqueous sodium carbonate solution and thendried over anhydrous sodium sulphate and evaporated to dryness. Theresidue was purified by flash chromatography (SiO2/ethyl acetate):cyclohexane {fraction (1/9)}). 1.25 g of7-hydroxy-6-dehydroestrone-3,7-diacetate (XIII)(3.41 mmol, 97%) wereobtained.

EXAMPLE 11

[0109] 7-Hydroxyestrone 3,7-diacetate (XIV)

[0110] A solution of 80 ml of glacial acetic acid containing 1.0 g of7-hydroxy-6-dehydroestrone-3,7-diacetate (XIII)(2.72 mmol) washydrogenated with 200 mg of 10% palladium on charcoal catalyst under ahydrogen pressure of 1 bar. The reaction medium was filtered after 2hours and evaporated to dryness. The residue was purified by flashchromatography (SiO2/ethyl acetate:cyclohexane {fraction (1/9)}). 855 mgof 7-hydroxyestrone 3,7-diacetate (XIV) (2.31 mmol, 85%) were obtained.

EXAMPLE 12

[0111] 7β-Hydroxyoestrone (XV)

[0112] A solution of 50 ml of methanol containing 1% of potassiumhydroxide and 1 g of 7-hydroxyestrone-3,7-diacetate (XIV)(2.70 mmol) washeated to reflux for 2 hours. The reaction medium was then cooled,neutralised and then extracted with ethyl acetate. The organic phase wasdried over anhydrous sodium sulphate and then evaporated to dryness. 695mg of 7β-hydroxyoestrone (XV)(2.43 mmol, 90%), which was recrystallisedfrom methanol, were obtained.

EXAMPLE 13

[0113] 70β-Hydroxyoestradiol (XVI)

[0114] 264 mg of sodium borohydride (7.00 mmol 2 eq.) were added to asolution of 50 ml of methanol containing 1.0 g of 7β-hydroxyoestrone(XV)(3.50 mmol). The reaction medium was poured into water and extractedwith ethyl acetate. The organic phase was dried over anhydrous sodiumsulphate and then evaporated to dryness. 917 mg of 7β-hydroxyoestradiol(XVI)(3.18 mmol, 91%), which was recrystallised from methanol, wereobtained.

EXAMPLE 14

[0115] DHEA-3-acetate (XVIII)

[0116] A solution of 50 ml of pyridine and 50 ml of acetic anhydridecontaining 10 g of DHEA (XVII)(34.72 mmol) was heated to reflux for 4hours. The reaction medium was cooled, poured into water and extractedwith ethyl acetate. The organic phase was dried over anhydrous sodiumsulphate and evaporated to dryness. 11.0 g of DHEA-3-acetate(XVIII)(33.33 mmol, 96%), which was recrystallised from ethanol, wereobtained.

EXAMPLE 15

[0117] 17-Ketal-DHEA-3-acetate (XIX)

[0118] A solution of 100 ml of toluene containing 5 g of DHEA-3-acetate(XVIII) (15.15 mmol), 5 ml of ethylene glycol and a catalytic amount ofp-toluenesulphonic acid was heated to reflux with steam distillationusing a Dean-Stark apparatus for 24 hours. The reaction medium waspoured into 100 ml of a 10% w/v aqueous potassium carbonate solution.The organic phase was decanted. The aqueous phase was extracted withethyl acetate. The organic phases were combined and evaporated todryness. 5.10 g of 17-ketal-3-DHEA-acetate (XIX)(13.64 mmol, 90%), whichwas recrystallised from ethanol, were obtained.

EXAMPLE 16

[0119] 7-Keto-17-ketal-DHEA-3-acetate (XX)

[0120] A solution of 70 ml of pyridine containing 5 g of17-ketal-DBEA-3-acetate (XIX)(13.37 mmol) and a catalytic amount ofBengal Rose was irradiated using a medium-pressure mercury vapour lampwith oxygen sparging. A catalytic amount of copper acetate was added tothe reaction medium after 24 hours. The reaction medium, after 24 hours,was evaporated to dryness. The residue was purified by flashchromatography (SiO2/ethyl acetate: cyclohexane {fraction (3/7)}). 3.11g of 7-keto-17-ketal-DHEA-3-acetate (XX)(8.02 mmol, 60%) were obtained.

EXAMPLE 17

[0121] 7-Keto-17-ketal-DHEA (XX)

[0122] A solution of 50 ml of methanol containing 1% of potassiumhydroxide and 1 g of 7-keto-17-ketal-DHEA-3-acetate (XX)(2.58 mmol) washeated to reflux for 2 hours. The reaction medium was then cooled,neutralised and then extracted with ethyl acetate. The organic phase wasdried over anhydrous sodium sulphate and then evaporated to dryness. 802mg of 7-keto-17-ketal-DHEA (XXI)(2.32 mmol 90%), which wasrecrystallised from methanol, were obtained.

EXAMPLE 18

[0123] 7-Hydroxy-17-ketal-EPIA (XXII)

[0124] 10 g of 7-keto-17-ketal-DHEA (XXI)(28.90 mmol) were added to aliquid ammonia solution at −33° C. containing 2.65 g of sodium. After 4hours, ammonium chloride was added until the blue colour disappeared.2.65 g of sodium were then added. After 4 hours, ammonium chloride wasagain added until the blue colour disappeared. Water was added and theammonia was allowed to evaporate. The reaction medium was extracted withethyl acetate. The organic phase was dried over anhydrous sodiumsulphate and then evaporated to dryness. 6.07 g of7-hydroxy-17-ketal-EPIA (XXII)(17.34 mmol, 60%) were obtained.

EXAMPLE 19

[0125] 7-Hydroxy-EPIA (XXIII)

[0126] A solution of 100 ml of acetone containing 5 ml of water, 10 g of7-hydroxy-17-ketal-EPIA (XXII)(28.57 mmol, 50%) and a catalytic amountof paratoluenesulphonic acid was heated to reflux for 4 hours. Thereaction medium was cooled, poured into 100 ml of a 10% w/v aqueoussodium carbonate solution and then extracted with ethyl acetate. Theorganic phase was dried over anhydrous sodium sulphate and thenevaporated to dryness. The residue was purified by flash chromatography(SiO2/ethyl acetate). 5.24 g of 7-hydroxy-EPIA (XXIII)(17.14 mmol, 60%)were obtained.

EXAMPLE 20

[0127] 70α-Hydroxy-EPIA (XXIV) & 7β-hydroxy-EPIA (XXV) 7-Hydroxy-EPIA(XXIII)(5 g) containing 7α and 7β epimers in a ratio 65/35 was purifiedby flash chromatography (Al₂O₃/CHCl₃). 7β-Hydroxy-EPIA (XXV)(2.5 g) wasobtained first, before 7α-hydroxy-EPIA (XXIV)(1.34 g). 7β-Hydroxy-EPIA(XXV) and 7α-hydroxy-EPIA (XXIV) were recrystallised from ethyl acetate.

EXAMPLE 21

[0128] Protocol For Studying Hypoxic Neuronal Damage

[0129] Organotypic hippocampal slice cultures were prepared using thebasic method of Pringle et al (1996, 1997) modified as follows:

[0130] Wistar rat pups (8-11 days old) were decapitated and thehippocampus rapidly dissected into ice-cold Gey's balanced salt solutionsupplemented with 4.5 mg/ml glucose. Slices were separated and platedonto Millicell CM culture inserts (4 per well) and maintained at 37°C./5% CO₂ for 14 days. Maintenance medium consisted of 25%heat-inactivated horse serum, 25% Hank's balanced salt solution (HBSS)and 50% minimum essential medium with added Earle's salts (MEM)supplemented with 1 mM glutamine and 4.5 mg/ml glucose. Medium waschanged every 3-4 days.

[0131] Experimental hypoxia was performed as described previously(Pringle et al., 1996; 1997). Briefly, cultures were transferred toserum free medium (SFM-75% MEM, 25% HBSS supplemented with 1 mMglutamine and 4.5 mg/ml glucose) containing 5 μg/ml of the fluorescentexclusion dye propidium iodide (PI). Cultures were allowed toequilibrate in SFM for 60 minutes prior to imaging. PI fluorescence wasdetected using a Leica inverted microscope fitted with a rhodaminefilter set. Any cultures in which PI fluorescence was detected at thisstage were excluded from further study. Hypoxia was induced bytransferring cultures to SFM (+PI) which had been saturated with 95%N₂/5% CO₂. Culture plates (without lids) were then sealed into anairtight chamber in which the atmosphere was saturated with 95% N₂/5%CO₂ by continuously blowing through gas at 10 litres/minute for tenminutes before being sealed and placed in the incubator for 170 minutes(total time of hypoxia was therefore 180 minutes). At the end of thehypoxic period cultures were returned to normoxic SFM containing PI andplaced back in the incubator for 24 hours.

[0132] Neuronal damage was assessed as described previously (Pringle etal., 1996; 1997) using either NIH Image 1.60 running on an Apple IIsicomputer or OpenLab 2.1 (Improvision) running on a Macintosh G4/400.Images were captured using a monochrome camera and saved onto opticaldisk for offline analysis. Light transmission images were captured priorto the addition of drugs, and PI fluorescence images recorded at the endof the 24-hour post-hypoxia recovery period. The area of the CA1 celllayer was determined from the transmission image. The area of PIfluorescence in CA1 was measured using the density slice function withinNIH Image or OpenLab, and neuronal damage expressed as the percentage ofthe CA1 in which

[0133] Steroid compounds were prepared by making an initial 1 mg/mlsolution in ethanol and further diluting down in SFM. Compounds wereadded to the cultures for 45 minutes prior to hypoxia, during thehypoxic episode and during the post-hypoxic recovery period. Controlexperiments consisted of cultures treated with vehicle alone.

RESULTS

[0134] Experiment 1:

[0135] An initial experiment was performed to determine whether7αOH-EPIA and 7βOH-EPIA were neuroprotective at a high concentration of100 nM. Hypoxia produced a lesion in 25.5±6.4% of CA1. This damage wassignificantly reduced by both 7αOH-EPIA and 7βOH-EPIA when present pre-,during and post-hypoxia (see Table I). TABLE I Compound N % Damage inCA1 Control Hypoxia 17 25.5 ± 6.4  Hypoxia + 100 nM 7αOH-EPIA 16  4.0 ±2.9** Hypoxia + 100 nM 7βOH-EPIA 16  9.0 ± 4.7*

[0136] Experiment 2:

[0137] Having determined that both the α and β-isomers of 7OH-EPIA wereneuroprotective, we assessed the concentration-dependency of thiseffect. Control hypoxia resulted in neuronal damage to 31.9±4.7% of theCA1. 7βOH-EPIA was significantly neuroprotective at 10 nM and 100 nM,but activity was lost if the concentration was reduced to 1 nM. as shownin Table II, below. TABLE II Compound N % Damage in CA1 Control Hypoxia29 31.9 ± 4.7  Hypoxia + 1 nM 7βOH-EPIA 15 20.6 ± 7.2  Hypoxia + 10 nM7βOH-EPIA 12 11.9 ± 4.7* Hypoxia + 100 nM 7βOH-EPIA 13 14.3 ± 5.0*

[0138] Experiment 3:

[0139] Having observed the neuroprotective activity of 7βOH-EPIA, wenext investigated whether 7βOH-DHEA was neuroprotective. Cultures wereincubated with either 100 nM 7βOH-DHEA or vehicle, pre-, during andpost-hypoxia. Hypoxia produced damage in 29.0±6.2% of CA1. In culturestreated with 7βOH-DHEA, a large, highly significant, reduction inneuronal damage was observed as shown in Table III, below. TABLE IIICompound N % Damage in CA1 Control Hypoxia 21 29.0 ± 6.2  Hypoxia + 100nM 7βOH-DHEA 16  4.2 ± 1.9**

EXAMPLE 22

[0140] Global Cerebral Ischaemia in Rats (4 Vessel Occlusion)

[0141] Cerebral ischaemia was induced by four-vessel-occlusion (4VO) inmale Wistar rats (250-280 g). Both vertebral arteries were occluded byelectrocauterization in pentobarbital anaesthesia (60 mg/kg i.p.). Theanimals were allowed to recover for 24 hours with free access to waterbut not food. The next day the carotid arteries were exposed under 2%halothane in 30% oxygen/70% nitrous oxide anaesthesia and were occludedfor 10 minutes using microvascular clamps. Subsequently, both clampswere removed and both arteries were inspected for immediate reperfusion.During the operation and the following 3 hours normothermia of theanimals (37.5±0.5° C.) was maintained by using a thermostaticallycontrolled heating blanket connected to a rectal thermometer. Forcontrol, in sham-operated animals both vertebral arteries werecauterised in pentobarbital anaesthesia and both common carotid arterieswere exposed but not clamped under 2% halothane in 30% oxygen/70%nitrous oxide anaesthesia the following day. The wound was treated withlidocaine gel and then sutured. The animals were kept under a heatinglamp at 30° C. environmental temperature until they regainedconsciousness.

[0142] Seven groups of animals were investigated:

[0143] 1. (n=8) steroid compound, 7β-OH EPIA (0.1 mg/kg, i. v. via tailvein, three injections: 15 minutes prior to the induction of ischaemia,during ischaemia and 5 minutes after reperfusion);

[0144] 2. (n=8) steroid compound, 7β-OH EPIA (0.3 mg/kg, i. v. threeinjections as described in 1.);

[0145] 3. (n=8) steroid compound, 7β-OH EPIA (1 mg/kg, i. v., threeinjections as described in 1.);

[0146] 4. (n=8) NBQX (disodium salt, because more water soluble) asreference substance and positive control (TOCRIS, Germany, 30 mg/kg, i.p., three injections as described in 1.);

[0147] 5. (n=8) received vehicle (0.9% NaCl, containing 100 μl Ethanol)three injections as described in 1.);

[0148] 6. (n=8) ischaemia alone;

[0149] 7. (n=8) sham operated controls.

[0150] NBQX was 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxalineand was known to have neuroprotective activity [Gill, R., Nordholm, L.,Lodge D.: The neuroprotective action of2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) in a ratfocal ischaemia model. Brain Res. 580, 35-43, 1992].

[0151] 7β-OH EPIA was 7β-hydroxyepiandrosterone, a compound of thepresent invention.

[0152] The substances were dissolved in 100 μl ethanol and finallydiluted with 0.9% NaCl.

[0153] After a survival time of 7 days after ischaemia, all animals wereperfusion fixed transcardially with 4% paraformaldehyde. The brains werethen removed carefully and postfixed in the same fixative for 2 hours.After cryoprotection in 30% sucrose, the brains were rapidly frozen inisopentane and stored at −80° C. Twenty-micrometer cryostat sectionscomprising the hippocampal formation were Nissl stained with toluidineblue or NeuroTrace fluorescence.

[0154] Data Analysis:

[0155] The severity of neuronal damage in the hippocampal CA1 regionafter ischaemia was evaluated by the number of surviving neurons usingNissl staining. The mean number of morphologically intact neurons per400 μm length was calculated in CA1 region for each group. Cell countingwas performed in 3-5 serial sections per animal and 6 times 400 μm CA1area per section using a light microscope equipped with a 20× objective.The data were statistically analysed by paired Student's t-test. Datawere presented as mean ± SEM.

[0156] Results and Discussion

[0157] Morphological intact hippocampal CA1 neurons were characterisedby Nissl staining (toluidine blue and NeuroTrace) with the followingcriteria: clear shape of a neuronal perikarya, large nucleus with apositive labelled nucleolus, a small cytoplasm zone around the nucleuswith positive Nissl staining, indicating the intact rough endoplasmicreticulum with ribosomes and therefore the intact protein synthesismachinery.

[0158] 10 minutes of global ischaemia (mild ischaemia) and a survivaltime of 7 days leads to a neurodegeneration of pyramidal cellsselectively in the hippocampal CA1 region (FIG. 1A-1C). The mean numberof pyramidal cells in CA1 of sham operated animals was 121.5±4.3 (set as100%). Therefore, 60% of CA1 neurons died after 10 minutes of globalischaemia (FIG. 1B). The number of neurons in the animal group ofischaemia and i. v. injection of vehicle (NaCl plus 100 μl Ethanol)applied as described in the experiment was comparable to that of theischaemia group alone (FIG. 1A, 1B). NBQX (30 mg/kg, i.v., threeinjections as described in the experiment) showed a significant (p=0.03)neuroprotection in CA1 pyramidal cells compared to the ischaemia group.Compared to the ischaemia alone NBQX leads to a 47.5% neuroprotectionwhile compared to the sham operated animals the protective effect was68.5%. The neuroprotection caused by NBQX was in agreement with Gill etal., 1992 and Gill 1994 demonstrating the validity of the globalischaemia model we used in our experiments. 7β-OH EPIA leads to aconcentration dependent neuroprotection of hippocampal CA1 pyramidalcells after 10 minutes of global ischaemia and a survival time of 7 days(FIG. 1A). T-test analysis revealed a highly significant neuroprotectiveeffect of 7β-OH EPIA in concentrations of 0.1 mg/kg (p=0.01) and 0.3mg/kg (p=0.0008). Compared to the sham operated group 7β-OH EPIA showeda 74.8% (0.1 mg/kg) and a 83.9% (0.3 mg/kg) neuroprotective effect onCA1 pyramidal cells, respectively (FIG. 1C). 7β-OH EPIA in aconcentration of 1.0 mg/kg showed only a tendency to neuroprotection,but the effect was not significant.

[0159] In all experiments with 7β-OH EPIA injected i.v. prior, duringand after ischaemia we never observed any behavioural abnormalities ofthe animals.

[0160] Legends of the Figures:

[0161] Number of morphological intact hippocampal CA1 pyramidal cells inrats 7 days after global cerebral ischaemia in rats and under theinfluence of different compounds.

[0162]FIG. 1A: Data were presented as mean number ± SEM of intactneurons per 400 μm length of CA1 region.

[0163]FIG. 1B: Data were expressed as percentage of intact neurons per400 μm length of CA1 region compared to sham operated animals set as100%.

[0164]FIG. 1C: Data were presented as absolute percentage ofneuroprotection when the number of surviving neurons in the ischaemiagroup was set to zero and those of the sham operated group was set to100%.

[0165] Although the above data demonstrate neuroprotection, it hasrecently been shown that Cyp7b1, the enzyme responsible for7-hydroxylation of 3-hydroxy steroids and which thus results in theconversion of oestradiol, DHEA and EPIA to their neuroprotectivederivatives, is present in other tissues subject to ischaemic injury,including cardiac and renal tissues. Accordingly, it can be deduced fromthe above data that the compounds of the present invention would protectcardiac and renal tissues from ischaemic injury.

EXAMPLE 23

[0166] Cardioprotective Effect

[0167] The study was designed to test the potential anti-ischaemic andcardioprotective effect of 7β-OH EPIA in Langendorf perfused male rathearts. The end point for evaluation of protection in this model isinfarct size (protection against lethal injury, cell death). The infarctsize model used is based on a standardised ischaemic insult followed byreperfusion. In the current study, treatment was added ex vivo (to theperfusion solution of the isolated heart) for 30 minutes prior toinfarction and continued through 30 minutes regional ischaemia(infarction) and 120 minutes of reperfusion. Based on results from pilotstudies, a concentration of 100 nM was used and compared to vehicletreated hearts. The drug was dissolved in DMSO (vehicle) and the finalconcentration of DMSO in the perfusion solution was 1:10⁻⁶. The resultsof the study showed that the compound at a concentration of 100 nMreduced infarct size significantly from 46.3+/−2.49 to 14.4+/−1.22% ofthe ischaemic risk zone (p<0.001). Heart function in conjunction withdrug addition was also examined, and addition of 7β-OH EPIA by itselfdid not result in detectable changes in heart function. Hearts treatedwith 7β-OH EPIA showed a slight reduction in global left ventricularsystolic pressure parameters during regional ischaemia (p<0.05 at 25minutes regional ischaemia), which disappeared after reperfusion.

[0168] The study confirms, cardioprotective, anti-ischaemic propertiesof the compound at a concentration of 100 nM in the isolated perfused,male rat heart.

[0169] Animal Treatment

[0170] Animals were housed in the animal department of the University ofTroms and treated according to the guidelines formulated by the EuropeanConvention for the protection of vertebrate animals used forexperimental or other purposes. Supply of Wistar rats were from Harland(The Netherlands), and the rats stayed in the animal department for oneweek before experiments were started. Animal weight was restricted to240-380 g, and only male hearts were used. This corresponds to age60-120 days. On the day of the experiment, rats were transferred in afilter cabinet from the animal department to the laboratory. The ratswere then anaesthetised by pentobarbital injection intraperitoneally(50-75 mg/kg) and heparinized by 200 IU also intraperitoneally.

[0171] Perfusion

[0172] The hearts were rapidly (within 1-2 minutes) transferred to aperfusion set up using Krebs Henseleits bicarbonate buffer as theperfusion solution. Perfusate and heart were maintained at 37° C.

[0173] The Krebs Henseleits bicarbonate buffer consisted of: NaCl 118.5mM/litre  NaHCO₃ 25.0 mM/litre  KCl 4.7 mM/litre KH₂PO₄ 1.2 mM/litreMgSO₄ 1.2 mM/litre CaCl₂ 2.4 mM/litre glucose 11.1 mM/litre 

[0174] The buffer was equilibrated with a gas mixture of approximately5% CO₂ in O₂.

[0175] The perfusion pressure was 100 mm H₂O. A latex balloon wasmounted on the tip of polyvinyl tubing, connected to a pressuretransducer and inserted into the cavity of the left ventricle formeasurements of left ventricular pressure. The size of the balloon wasnot changed during the experiment and the experimental set up wastherefore an isovolumetric left ventricle preparation. Timed collectionsof venous effluate from the right side of the heart (sinus coronariusvia the pulmonary trunk and the right atrium) were used for coronaryflow measurements. Heart rate was calculated from pressure recordings.

[0176] Computer-based data acquisition and analysis were used for heartfunction (Lab View based software).

[0177] Experimental Protocol

[0178] The stabilisation period was 20-25 minutes, and hearts which didnot reach a stable performance during this period, were discharged.Hearts with a LVDP (left ventricular developed pressure) between 80 mmHgand 175 mmHg, diastolic pressure between 0-10 mmHg and coronary flowbetween 9 to 18 ml/min at the end of the stabilisation period were used.

[0179] A 100 mM stock solution of 7β-OH EPIA in DMSO was made and storedas aliquots in eppendorf tubes at 20° C. This solution was furtherdiluted in perfusion solution to 100 nM of active drug just prior to theexperiment. Pretreatments with 7β-OH EPIA (n=8) or vehicle (n=8) weregiven for 30 minutes before infarction and continued until the end ofexperiments.

[0180] Regional ischaemia (infarction) was achieved by placing a silksuture around the main branch of the left coronary artery. Reversibleligation was obtained by using a small piece of a polyvinyl tube. Thestandardised regional ischaemia lasted 30 minutes, and hearts were thenreperfused for 2 hours.

[0181] Infarct Size and Risk Zone Size

[0182] At the end of the experiment the following procedure wasfollowed:

[0183] 1) The left coronary artery was re-ligated

[0184] 2) A suspension of blue dye was added to the perfusion line. Theischaemic zone was identified as the area without dye.

[0185] 3) The heart was quickly removed, weighed and frozen.

[0186] 4) One day thereafter the heart was cut in 2 mm thick slices andstained with tetrazolium. The heart slices were incubated for 20 minutesin 1% TTC (triphenyl-tetrazolium- chloride) in 0.2 M phosphate buffer(pH 7.4) at 37° C. Infarcted tissue will not develop any colour reactionwhereas surviving tissue develops a purple colour due to the presence ofdehydrogenases and cofactors in the tissue.

[0187] 5) After staining, hearts were placed in formalin (4%) forfixation.

[0188] 6) TTC staining will be lost with time and all heart slices weretherefore computer scanned and saved as digital images fordocumentation.

[0189] 7) Computer based planimetry was used to calculate volume of theventricles, the ischaemic risk zone and infarct. Results are presentedas infarct in % of ischaemic risk zone.

[0190] Results

[0191] The results are shown in the following Table 1. TABLE 1 Bodyweight, heart weight, left ventricle volume, risk zone volume andinfarct volume (mean SEM) in the control and treated group. control (n =8) 7β-OH EPIA (n = 8) Rat weight (g) 295.6 +/− 5.30 285.6 +/− 7.93 heartweight (g)  1.3 +/− 0.04  1.3 +/− 0.04 ventricle volume (mm3) 239.0 +/−17.73 200.3 +/− 9.23 Risk zone volume (mm3) 109.3 +/− 8.43  98.9 +/−6.51 Infarct volume  38.9 +/− 4.98  14.1 +/− 1.46 * p < 0.001 Riskzone/ventricle (%)  35.7 +/− 3.11  49.3 +/− 2.38 Infarct/risk zone (%) 46.3 +/− 2.49  14.4 +/− 1.22 * p < 0.001

[0192] The results are also illustrated in FIGS. 2 to 6 of theaccompanying drawings.

[0193] Infarct:

[0194] There was a significant difference between the two groups(p<0.001) with a marked infarct reduction in the drug treated group.Infarcts in the control vehicle treated hearts were comparable in sizeto the standard control infarcts usually obtained in untreated heartssubjected to 30 minutes regional ischaemia

[0195] Heart Function:

[0196] Heart function was examined throughout the experimental timecourse. A marked decrease in coronary flow was seen in conjunction withthe coronary artery occlusion in this experimental model and confirmsthat the intended coronary occlusion took place. Coronary flow was notinfluenced by the addition of 7β-OH EPIA. Heart-rate was notsignificantly different between the two groups and remained stablethroughout the experiment. Left ventricular developed pressure (LVDP)also declines during regional ischaemia. This decline was larger in drugtreated hearts compared to the control. A significant difference in LVDPbetween the groups was seen at 25 minutes of regional ischaemia. Nodifferences between the two groups were observed in diastolic pressure.

[0197] Vehicle:

[0198] Vehicle DMSO has previously been used as a vehicle inbuffer-perfused hearts and the results indicate that under thecircumstances of the current study this compound has no detectableinfluence by itself.

[0199] Conclusion

[0200] The study demonstrated a marked and significant cardioprotectiveeffect of 7β-OH EPIA. The extent of protection is comparable inmagnitude to some of the most potent and well characterisedcardioprotective drugs or treatment regimes like NHE blockade orischaemic preconditioning when used in the same experimental model.

1. The use of a 3-hydroxy-7-hydroxy steroid or a 3-oxo-7-hydroxy steroidor a pharmaceutically acceptable ester thereof for the manufacture of amedicament for protection against ischaemic damage to peripheral organs,and for the treatment of spinal cord injury-induced damage to the spinalcord.
 2. The use according to claim 1, in which the steroid is acompound of formula (I):

wherein R¹ and R² are the same as or different from each other and eachrepresents a hydrogen atom, an alkyl group having from 1 to 6 carbonatoms, an alkenyl group having from 2 to 6 carbon atoms, an alkynylgroup having from 2 to 6 carbon atoms, an aryl group having from 6 to 10carbon atoms, a formyl group, an alkylcarbonyl group having from 2 to 7carbon atoms, an alkenylcarbonyl group having from 3 to 7 carbon atoms,an alkynylcarbonyl group having from 3 to 7 carbon atoms, anarylcarbonyl group having from 7 to 11 carbon atoms, an aralkylcarbonylgroup having from 8 to 15 carbon atoms, an aralkenylcarbonyl grouphaving from 9 to 15 carbon atoms, a residue of an amino acid, or aheterocyclic-carbonyl group, as defined below; one of R^(a) and R^(b)represents a group of formula —R^(c), preferably in the β configuration,and the other represents a hydrogen atom, or R^(a) and R^(b) togetherrepresent an oxo group; R^(c) represents an alkanoyl group having from 1to 6 carbon atoms, an aryl-carbonyl group, in which the aryl part was anaromatic carbocyclic group having from 6 to 10 ring carbon atoms, aheterocyclic-carbonyl group, as defined below, or a group of formula—OR⁴, where R⁴ represents ay one of the groups and atoms defined abovefor R¹ and R²; the ring A,

is a benzene or cyclohexane ring; when ring A is a cyclohexane ring, thedotted line in ring B represents a single or double carbon-carbon bondand n was 1; or when ring A is a benzene ring, the dotted line in ring Brepresents a single carbon-carbon bond and n is 0; saidheterocyclic-carbonyl group is a group of formula R³—CO, where R³represents a heterocyclic group having from 3 to 7 ring atoms, of whichfrom 1 to 3 are hetero-atoms selected from nitrogen atoms, oxygen atomsand sulphur atoms, and the remaining atom or atoms of which there is atleast one is or are carbon atoms; said alkyl, alkenyl and alkynyl groupsand the alkyl, alkenyl and alkynyl parts of said alkylcarbonyl,alkenylcarbonyl and alkynylcarbonyl groups being unsubstituted or havingat least one of the following substituents Ψ: substituents Ψ: hydroxygroups, mercapto groups, halogen atoms, amino groups, alkylamino groupshaving from 1 to, 6 carbon atoms, dialkylamino groups in which eachalkyl group has from 1 to 6 carbon atom, carbamoyl groups, nitro groups,alkoxy groups having from 1 to 6 carbon atoms, alkylthio groups havingfrom 1 to 6 carbon atoms, carboxy groups, alkoxycarbonyl groups andunsubstituted aryl groups having from 6 to 10 carbon atoms; said arylgroups, said heterocyclic groups, and the aryl parts of saidarylcarbonyl groups and said aralkylcarbonyl groups being unsubstitutedor having at least one of the following substituents ξ: substituents ξ:any of substituents Ψ, and alkyl groups having from 1 to 6 carbon atoms,hydroxyalkyl groups having from 1 to 6 carbon atoms, and haloalkylgroups having from 1 to 6 carbon atoms; and pharmaceutically acceptablesalts and esters thereof.
 3. The use according to Claim 2, in which: R¹and R² are the same as or different from each other and each representsa hydrogen atom, an alkyl group having from 1 to 6 carbon atoms, anoptionally substituted phenyl group, a formyl group, an alkylcarbonylgroup having from 2 to 5 carbon atoms, an arylcarbonyl group having from7 to 11 carbon atoms, an aralkylcarbonyl group having from 8 to 15carbon atoms, a residue of an amino acid, or a heterocyclic-carbonylgroup, as defined below; one of R^(a) and R^(b) represents an alkanoylgroup having from 1 to 6 carbon atoms or a group of formula —OR⁴, whereR⁴ represents any one of the groups and atoms defined above for R¹ andR², in the β configuration, and the other represents a hydrogen atom, orR^(a) and R^(b) together represent an oxo group; saidheterocyclic-carbonyl group is a group of formula R³—CO, where R³represents a heterocyclic group having from 3 to 7 ring atoms, of whichfrom 1 to 3 are hetero-atoms selected from nitrogen atoms, oxygen atomsand sulphur atoms, and the remaining atom or atoms of which there is atleast one is or are carbon atoms.
 4. The use according to claim 1, inwhich the steroid is a compound of formula (II):

in which: R² represents a hydrogen atom, an alkyl group having from 1 to6 carbon atoms, an optionally substituted phenyl group, a formyl group,an alkylcarbonyl group having from 2 to 5 carbon atoms, an arylcarbonylgroup having from 7 to 11 carbon atoms, an aralkylcarbonyl group havingfrom 8 to 15 carbon atoms, a residue of an amino acid, or aheterocyclic-carbonyl group, as defined below; one of R^(a) and R^(b)represents a group of formula R^(c), preferably in the β configuration,and the other represents a hydrogen atom, or R^(a) and R^(b) togetherrepresent an oxo group; R^(c) represents an alkanoyl group having from 1to 6 carbon atoms, an aryl-carbonyl group, in which the aryl part is anaromatic carbocyclic group having from 6 to 10 ring carbon atoms, aheterocyclic-carbonyl group, as defined below, or a group of formula—OR⁴, where R⁴ represents any one of the groups and atoms defined aboveR²; said heterocyclic-carbonyl group is a group of formula R³—CO, whereR³ represents a heterocyclic group having from 3 to 7 ring atoms, ofwhich from 1 to 3 are hetero-atoms selected from nitrogen atoms, oxygenatoms and sulphur atoms, and the remaining atom or atoms of which thereis at least one is or are carbon atoms, or a pharmaceutically acceptablesalt or ester thereof.
 5. The use according to any one of claims 2 to 4,in which the —OR² group at the 7-position is in the β-configuration. 6.The use according to claim 1, in which the steroid is7β-hydroxy-epiandrosterone.
 7. The use according to claim 1, in whichthe steroid is 7β-hydroxy-dehydro-epiandrosterone.
 8. The use accordingto claim 1, in which the steroid is 7β-hydroxy-17β-oestradiol.
 9. Theuse according to claim 1, in which the steroid is7β-hydroxy-pregnenolone.
 10. The use according to claim 1, in which thesteroid is 7β-hydroxy-oestrone.
 11. The use according to claim 1, inwhich the steroid is 7α-hydroxy-oestrone.
 12. The use according to anyone of the preceding claims, in which the peripheral organ is the heart.13. The use according to claim 12, in which the damage to the heart iscaused by a myocardial infarction.
 14. The use according to any one ofclaims 1 to 11, in which the peripheral organ is the kidneys.
 15. Theuse according to claim 14, in which the damage to the kidneys is causedby glomerulonephritis or acute renal failure.
 16. The use according toany one of claims 1 to 11, which is for the treatment of injury to thespinal cord.
 17. A method of protecting a mammal againstischaemia-induced tissue damage in peripheral organs or against spinalcord injury-induced damage to the spinal cord, by administering theretoan effective amount of a 3-hydroxy-7-hydroxy steroid or a3-oxo-7-hydroxy steroid or a pharmaceutically acceptable ester thereof,as defined in any one of claims 1 to
 11. 18. A method according to claim17, in which the peripheral organ is the heart.
 19. A method accordingto claim 17, in which the peripheral organ is the kidneys.
 20. A methodaccording to claim 17, in which the mammal is protected against damagecaused by injury to the spinal cord.